This project will use purified protein systems and cell-free extracts to test models for two steps of DNA interstrand crosslink (ICL) repair in mammalian cells. In G{1} phase or quiescent cells, models incorporate an unhooking step mediated by nucleotide excision repair (NER), with incisions on one strand both 5' and 3' of an ICL. There is conflicting published data on this subject, which this research aims to resolve. A critical aspect of ICL repair models is that an unhooked ICL can be bypassed in a REV3L-dependent manner. Assays for both of these reactions will be carried out in cell extracts, including those defective in specific DNA repair factors. Normal cell extracts will be fractionated to determine which factors are necessary for the full reactions, and fractions substituted with purifed protein components, to recapitulate and reconstitute these processes. Specific Aim 1 is to define the positions of unhooking of an ICL by nicking on both sides, and examine open complex formation by human cell extracts. Our unpublished data indicates that mammalian cell extracts can cleave both 5' and 3' of a preferentially acting on the pyrone side of the crosslink. This aim will be carried out in collaboration with Project 2. Specific Aim 2 will identify the protein factors required for ICL unhooking. Experiments will examine the genetic dependence of the reaction by testing cell lines with specific defects in factors from these pathways. Further, extracts will be fractionated and unhooking of an ICL reconstituted with purified proteins. Specific Aim 3 is to test final stages of ICL repair models. We will determine whether specialized DNA polymerases can bypass an unhooked ICL and how an unhooked ICL can be removed in duplex DNA. The genetic dependence of synthesis over an unhooked ICL will be examined using cell extracts and purified proteins. Proteins to be employed include REV3, REV7, REV1 and other DNA polymerases. The final step in ICL repair will be examined, using a substrate representing an unhooked psoralen crosslink attached to duplex DNA. The ability of NER to cleave this substrate will be analyzed, and compared to excision by BER.